JP4566494B2 - Methods for CpG receptor (CpG-R) and CpG-R - Google Patents

Abstract

The present invention is directed to nucleic acid molecules and polypeptides encoding a CpG receptor (CpG-R). The CpG-R contains a THD, interacts with the MyD88 adapter protein, and may bind to CpG oligonucleotides. The present invention is also directed to antibodies against CpG-R and to methods of modulating an immune response and to methods of identifying compounds which bind to and/or modulate CpG-R.

Description

【００４９】
。
【００５０】
（イムノブロッティング）
刺激後、完全Ｊプロテアーゼインヒビターカクテル（Ｃｏｍｐｌｅｔｅ Ｊ ｐｒｏｔｅａｓｅ ｉｎｈｉｂｉｔｏｒ ｃｏｃｋｔａｉｌ）（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ）（Ｔｒｉｔｏｎ ｌｙｓｉｓ ｂｕｆｆｅｒ）とともに、１％Ｔｒｉｔｏｎ Ｘ−１００，５０ｍＭ Ｔｒｉｓ，６２．５ｍＭ ＥＤＴＡ（ｐＨ８．０）中で、ＢＭＭＯまたはＲＡＷ ２６４．７細胞を溶解した。溶解物を、還元サンプル緩衝液中で煮沸し、ＮｕＰＡＧＥＪ Ｂｉｓ−Ｔｒｉｓ電気泳動システム（Ｎｏｖｅｘ）を用いて、１０％ポリアクリルアミドゲル上で分離した。製造業者の指示に従って、ＩκＢ−α、リン酸化−ＩκＢ−α（Ｎｅｗ Ｅｎｇｌａｎｄ ＢｉｏＬａｂｓ）、または抗ＩＬ−１８（Ｓａｎｔａ Ｃｒｕｚ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ）に対する抗体を用いて、ニトロセルロースメンブレンをプローブして、強化化学発光（Ａｍｅｒｓｈａｍ）を用いて、可視化した。
【００５１】
（κＢ−Ｌｕｃアッセイ）
トランスフェクションの２４時間前に、１ウェルあたり、３×１０⁵細胞の密度で、ＲＡＷ ２６４．７細胞を６ウェルプレート（Ｃｏｒｎｉｎｇ）に播種した。トランスフェクションにおいて用いたプラスミドは、ｐＮＦ−κＢ−Ｌｕｃ（Ｃｌｏｎｔｅｃｈ）およびｐＣＲ３．Ｖ６４−Ｍｅｔ−Ｆｌａｇ−ＭｙＤ８８ｌｐｒ（Ｊｕｒｇｅｎ Ｔｓｃｈｏｐｐより寄贈；Ｂｕｒｎｓら、Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．１９９８，２７３，１２２０３〜９に記載）であった。空のベクター（ｐＣＭＶＫｍ２、Ｃｈｉｒｏｎ）の添加により、全てのウェルにまたがって、総プラスミド濃度を正規化した。製造業者の指示に従って、１ウェルあたり、１０ｍｌのＬｉｐｏｆｅｃｔＡＭＩＮＥ（Ｇｉｂｃｏ ＢＲＬ）とともに、言及された濃度のＤＮＡ含有Ｏｐｔｉ−ＭＥＭ Ｉ（Ｇｉｂｃｏ ＢＲＬ）を用いて細胞をトランスフェクトした。細胞を３７℃で３時間トランスフェクション混合液とともにインキュベートし、次いで、培養培地を置換し、そして細胞を一晩、回収させた。翌日、示された濃度および時間で、ホスホロチオエートオリゴヌクレオチド、ＭＰＬ、ＬＰＳまたはサイトカインを用いて、３７℃、５％ＣＯ₂で、培養培地中で、トランスフェクトした細胞を、処理した。冷リン酸緩衝化生理食塩水（ＰＢＳ）を用いて細胞を１回洗浄し、そしてＲｅｐｏｒｔｅｒ Ｌｙｓｉｓ Ｂｕｆｆｅｒ Ｊ（Ｐｒｏｍｅｇａ）を用いて溶解した。全て、製造業者の指示に従って、Ｍｉｃｒｏｌｉｔｅ ２プレート（Ｄｙｎｅｘ）およびＭＬ ３０００ Ｌｕｍｉｎｏｍｅｔｅｒ（Ｄｙｎａｔｅｃｈ）を用いて、溶解上清中のルシフェラーゼ活性を決定した。ＦＬＡＧ−ＭｙＤ８８ｌｐｒ発現の検出のために、トランスフェクトされたＲＡＷ ２６４．７細胞由来の可溶性ペレットをＴｒｉｔｏｎ溶解緩衝液中に再懸濁し、そして１０分間超音波処理し、その後、サンプル緩衝液を添加して、煮沸した。分離およびニトロセルロースメンブレンへの転写後、ＦＬＡＧ Ｍ２（Ｓｉｇｍａ）抗体を用いてメンブレンをイムノブロットした。
【００５２】
（フローサイトメトリー）
ＢＭＤＤＣを、示されたように、アジュバントを用いて一晩処理し、洗浄し、ＦｃＢｌｏｃｋ（０．２５μｇ／１０⁶細胞；Ｐｈａｒｍｉｎｇｅｎ）含有、冷ＰＢＳ／２％ ＦＢＳ中に再懸濁し、そしてＦＩＴＣ結合体化抗体およびＰＥ結合体化抗体（１μｇ／１０⁶細胞）を５分後に添加した。細胞を３０分間氷上で抗体とともにインキュベートし、洗浄し、そしてＦＡＣＳｃａｎ（Ｂｅｃｔｏｎ−Ｄｉｃｋｅｎｓｏｎ）上でフローサイトメトリーによって分析した。この分析には、ＣＤ１１ｃについてポジティブに染色し、かつ生存細胞の前方散乱および側方散乱の特性を有するＢＭＤＤＣ培養物由来の細胞のみが含まれた。アジュバントで処理した生存ＣＤ１１ｃ細胞のＣＤ８６−ＦＩＴＣ染色の幾何平均蛍光が未処理のＢＭＤＤＣ培養の幾何平均ＣＤ８６−ＦＩＴＣ蛍光を正規化することに基づいて、ＣＤ８６発現の変化を、評価する。
【００５３】
（実施例２：ＣｐＧオリゴヌクレオチドは、ＭＡＰＫ経路およびＮＦ−κＢを活性化する）
ＣｐＧモチーフを含むかまたは欠くオリゴヌクレオチドに対する、マウスＢＭＭＯおよび樹状細胞ならびにマウスマクロファージ細胞株ＲＡＷ ２６４．７のいくつかの生化学的反応を比較した。オリゴヌクレオチドにおけるＣｐＧモチーフの効果についてのモデルとして、１８２６と呼ばれるオリゴヌクレオチド（Ｃｈｕら、Ｊ．Ｅｘｐ．Ｍｅｄ．，１９９７，１８６，１６２３〜３１）（マウスにおける、その活性は文献中に広範に報告されている）を用いた（Ｂａｃｈｍａｉｅｒら、Ｓｃｉｅｎｃｅ，１９９９，１８３，１３３５〜９）。ＣｐＧおよびコントロールオリゴヌクレオチドを、ＤＮＡの安定性を強化するホスホロチオエート骨格を用いて各２０マーに合成した（Ａｇｒａｗａｌら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，１９９１，８８，７５９５〜９）。
【００５４】
実施例１に記載のように、Ｃ５７Ｂｌ／６マウスの骨髄細胞から、マクロファージを分化し、次いで、Ｍ−ＣＳＦを欠く低血清培地に４時間トランスファーした。培地単独、ホスホロチオエート改変ＣｐＧオリゴヌクレオチド（１μＭ）、ＣＧ配列を欠くコントロールオリゴヌクレオチド（１μＭ）、またはＭＰＬ（１００ｎｇ／ｍｌ）で３０分間処理した細胞から、溶解物を調製して、リン酸化ＥＲＫ１を認識する抗体を用いて、イムノブロッティングを実施し、そしてＥＲＫ．２ＲＡＷ ２６４．７マクロファージをＬＰＳ（１００ｎｇ／ｍｌ）でのさらなる処理とともに１５分間同様に処理して、そしてリン酸化ＥＲＫ１およびＥＲＫ２についてイムノブロッティングした。ＡＰＣまたはＲＡＷ ２６４．７細胞由来の骨髄からの全細胞抽出物のイムノブロッティングによって、ＣｐＧオリゴヌクレオチドによる複数のマイトジェン活性化プロテインキナーゼ（ＭＡＰＫ）経路（ＥＲＫ１／２，ＪＮＫおよびｐ３８を含む）の活性化が実証された。３つの細胞型全てにおいて、ＣｐＧオリゴヌクレオチドでの刺激の１時間以内に、ＥＲＫ、ＪＮＫおよび／またはＪＮＫ基質ｃ−ｊｕｎのリン酸化、およびｐ３８ ＭＡＰＫ基質ＡＴＦ ２のリン酸化を観察したが、ＣｐＧ配列を欠くコントロールでは観察しなかった（データ示さず）。これらの結果は、ＣｐＧオリゴヌクレオチドによるＪＮＫ経路およびｐ３８経路の迅速な活性化を示す。ＲＡＷ ２６４．７細胞およびＢＭＭＯにおけるＥＲＫ１／２ ＭＡＰＫ活性化についてのポジティブな結果は、別のマウスマクロファージ細胞株Ｊ７７４における観察に対して対照的であるが、ＥＲＫリン酸化は、ＣｐＧオリゴヌクレオチド処理細胞においては検出されなかった。
【００５５】
ＣｐＧオリゴヌクレオチドおよびコントロールオリゴヌクレオチドを含む、パネルのアジュバントで処理した、ＢＭＭＯおよびＲＡＷ ２６４．７細胞におけるＮＦ−κＢ経路の状態をまた試験した。ＬＰＳ（１μｇ／ｍｌ）、ＭＰＬ（１μｇ／ｍｌ）ホスホロチオエート改変ＣｐＧオリゴヌクレオチド（４μＭ）またはコントロールホスホロチオエートオリゴヌクレオチド（非ＣｐＧ；４μＭ）を用いて、ＢＭＭＯを１．５時間処理した。全細胞溶解物を調製し、そして上記のようにＩκＢαについてイムノブロッティングした。ＬＰＳ、ＭＰＬまたはＣｐＧオリゴヌクレオチドで処理したＢＭＭＯにおいて、１．５時間内にＮＦ−κＢ阻害性タンパク質ＩκＢαの分解を観察したが、コントロールオリゴヌクレオチドでは観察しなかった（データ示さず）。
【００５６】
また、未処理のＲＡＷ ２６４．７細胞またはホスホロチオエートＣｐＧオリゴヌクレオチド（４μＭ）で、５分、１０分、２０分および６０分、ならびにＣｐＧモチーフを欠くコントロールオリゴヌクレオチドで６０分間、処理した細胞から、溶解物を調製した。次いで、この溶解物を電気泳動によって分離し、そしてリン酸化ＩκＢαおよび総ＩκＢαに対する抗血清を用いてイムノブロッティングした。ＩκＢαリン酸化およびＲＡＷ ２６４．７細胞における分解により、ＣｐＧオリゴヌクレオチドによる類似の特異的活性化が示された。この活性化は、ＣｐＧオリゴヌクレオチド添加の１０分間内にＩκＢαリン酸化のレベルで検出され、そしてＩκＢαの分解は２０分間で顕著であった。ＩκＢαは、１時間内にＣｐＧオリゴヌクレオチド処理ＲＡＷ ２６４．７細胞中で再合成されたが、ほとんどのＩκＢαはリン酸化され、このことは迅速な分解についての連続的標的化を示唆する（データ示さず）。
【００５７】
ＣｐＧオリゴヌクレオチドまたはＭＰＬ処理により誘導されたκＢ依存性転写を直接評価するため、κ軽鎖エンハンサー（κＢ−Ｌｕｃ）のマルチコピーの制御下で、ルシフェラーゼをコードするＮＦ−κＢ−誘導性レポータープラスミドを用いて、ＲＡＷ２６４．７マクロファージをトランスフェクトし、そして翌日アジュバントまたはサイトカインを用いて処理した。詳細には、κＢ駆動ルシフェラーゼレポーター遺伝子を用いて、ＲＡＷ ２６４．７細胞をトランスフェクトし、そして２４時間後に、ＩＬ−１（２０ｎｇ／ｍｌ）、ＭＰＬ（１μｇ／ｍｌ）、ＬＰＳ（１μｇ／ｍｌ）またはホスホロチオエートＣｐＧオリゴヌクレオチド（４μＭ）を用いて６時間処理した。細胞溶解物中のルシフェラーゼ活性を照度計（ｌｕｍｉｎｏｍｅｔｒｙ）によって定量し、そして未処理のトランスフェクト細胞におけるルシフェラーゼシグナルに対してこの結果を正規化した。図１Ａに示されるように、ＲＡＷ ２６４．７細胞に対するＣｐＧオリゴヌクレオチドの添加は、ＭＰＬまたはＬＰＳにより誘導されたレベルと類似のレベルまで、κＢ依存性ルシフェラーゼ発現を誘導した。ＣｐＧオリゴヌクレオチドによるκＢレポーター遺伝子の活性化は、ＭＰＬまたはＬＰＳ（両方ともマクロファージにおいてＮＦ−κＢの強力なアクチベータ）に対する応答において見られる活性化よりもわずかに弱かった。ＩＬ−１は、ＩＬ−１８がそうであるように、ＲＡＷ ２６４．７細胞（図１Ａ）において、κＢ−Ｌｕｃの弱いアクチベータであった（データ示さず）。
【００５８】
κＢ依存性ルシフェラーゼ発現に対するＣｐＧオリゴヌクレオチドの効果は、用量依存性および時間依存性であった（図１Ｂおよび図１Ｃを参照のこと）。図１Ｂに関して、κＢ−ルシフェラーゼレポータープラスミドで一時的にトランスフェクトされたＲＡＷ ２６４．７細胞を、０．２５μＭ〜８μＭの範囲にわたるホスホロチオエートＣｐＧオリゴヌクレオチド濃度の濃度で、またはコントロールのオリゴヌクレオチド（４μＭ）で２４時間後に６時間にわたって処理した。活性化は、０．２５μＭ程度の低いＣｐＧオリゴヌクレオチド濃度で検出された（図１Ｂ）が、試験した最低濃度（０．０５μＭ）では検出されなかった（データは示さず）。図１Ｂに示すデータは、同じ６ウェル組織培養プレート上の未処理のトランスフェクトしたウェルからの溶解産物におけるルシフェラーゼレベルに対して正規化した、２連のウェルからの溶解産物における平均ルシフェラーゼ活性である。ＣｐＧオリゴヌクレオチド処理によるレポーター遺伝子の活性化は、８μＭで最大であり（図１Ｂ）、そして１０μＭの用量ではさらには増強されなかった（データは示さず）。図１Ｃに関して、κＢ−Ｌｕｃで一時的にトランスフェクトし、そして２４時間後にＣｐＧ含有オリゴヌクレオチド（４μＭ；白丸）またはＬＰＳ（１μｇ／ｍｌ；黒丸）で処理したＲＡＷ ２６４．７細胞。図１Ｃに示す結果は、未処理のトランスフェクトされたウェルに対して正規化された２連のウェルの平均である。ＣｐＧモチーフを欠く類似の濃度のコントロールのオリゴヌクレオチドに対する、κＢ-ＬｕｃでトランスフェクトされたＲＡＷ ２６４．７細胞の暴露は、ルシフェラーゼ発現の増大を誘導しなかった（図１Ｂおよび図１Ｃ）。ＲＡＷ ２６４．７マクロファージにおけるκＢ依存性ルシフェラーゼ発現は、ＣｐＧオリゴヌクレオチドまたはＬＰＳで処理した細胞において類似の反応速度を示した（図１Ｃ）；両方の処理は、迅速な応答（２時間で、２倍の増大よりも大きい）を誘導し、この応答は、４時間〜６時間でプラトーに達した。
【００５９】
これらの結果は、ＲＡＷ ２６４．７細胞におけるＣｐＧオリゴヌクレオチドによるＮＦ−κＢ経路の活性化が、特異的であり、迅速であり、そして用量依存性であることを実証する。サイトカインによって媒介されるＣｐＧオリゴヌクレオチドの間接的効果は、シグナル伝達の反応速度が非常に迅速であることを考慮すると、ありそうもない。ＣｐＧオリゴヌクレオチド処理ＲＡＷ ２６４．７細胞からの上清の、κＢ−ＬｕｃトランスフェクトＲＡＷ ２６４．７マクロファージに対する添加は、ルシフェラーゼ発現をほとんど誘導せず（４時間目で２倍未満）、このことは、κＢ−Ｌｕｃ転写に対するＣｐＧモチーフが直接的な効果を有することを確認した。
【００６０】
（実施例３：ドミナントネガティブＭｙＤ８８の発現は、κＢ依存性レポーター遺伝子のＣｐＧ誘導をブロックする）
種々の細菌由来構造体（ＬＰＳ、ペプチドグリカンおよびリポタンパク質を含む）に応じたＴｏｌｌレセプターの役割を考慮すると、Ｔｏｌｌ関連レセプターは、非メチル化ＣｐＧ配列を含むオリゴヌクレオチドに応じたシグナル伝達に関与すると考えられる。非メチル化ＣｐＧ配列はまた、病原性の非自己の特徴である。このために、ＲＡＷ ２６４．７細胞を、κＢ−ルシフェラーゼプラスミドおよびドミナントネガティブ形態のＭｙＤ８８（Ｔｏｌｌ／ＩＬ−１レセプターファミリーのメンバーによるシグナル伝達のために必要とされるアダプタータンパク質）をコードする発現ベクターで同時トランスフェクトした。これらの実験において用いられるドミナントネガティブＭｙＤ８８であるＭｙＤ８８ｌｐｒは、インタクトなＴｏｌｌ相同性ドメインを有するが、細胞死ドメイン（ｄｅａｔｈ ｄｏｍａｉｎ；ＤＤ）に点変異を含む。ｌｐｒマウスにおけるＦａｓの細胞死ドメインにおける類似の変異は、恐らく、この細胞死ドメインのコンホメーションを変更することによってＦａｓシグナル伝達を無くし、従って、下流のシグナル伝達分子の会合をブロックする。ＲＡＷ ２６４．７細胞におけるＭｙＤ８８ｌｐｒの過剰発現は、Ｔｏｌｌ相同性ドメインを含むタンパク質との会合について内因性ＭｙＤ８８と競合することによって、ＭｙＤ８８依存性シグナル伝達を中断すると予想される。ＭｙＤ８８ｌｐｒのこの効果は、この死ドメイン（ｄｅａｔｈ）における変異が、ＮＦ−κＢの他のアクチベーターと共有され得る下流のシグナル伝達構成要素との会合を妨げるに違いないので、Ｔｏｌｌ関連タンパク質に対して特異的である。
【００６１】
ＦＬＡＧタグ化ＭｙＤ８８ｌｐｒをコードするプラスミドでのＲＡＷ ２６４．７細胞のトランスフェクションは、抗ＦＬＡＧ抗体での免疫ブロッティングによって決定した場合、予想された大きさのタンパク質の発現をもたらした（データは示さず）。図２Ａに関して、次いで、ＲＡＷ ２６４．７細胞を、κＢ−Ｌｕｃ（１μｇ）単独で（黒棒）またはＭｙＤ８８ｌｐｒをコードするプラスミドで、２つの異なる比率（１０：１（灰色の棒）または１：１（白色の棒））でトランスフェクトした。総プラスミドＤＮＡ濃度を、空のベクターの添加によって、全てのウェルにまたがって正規化した。トランスフェクション２４時間後の細胞を、ＣｐＧオリゴヌクレオチド（４μＭ）、ＣｐＧモチーフを欠くコントロールオリゴヌクレオチド（４μＭ）、ＭＰＬ（１μｇ／ｍｌ）またはＬＰＳ（１μｇ／ｍｌ）で処理した。示した結果は、未刺激のトランスフェクトされたコントロールの平均値（未処理カラム）に対して正規化された２連のウェルの平均＋／−標準偏差であり、そして複数の実験において得られた結果の代表である。ドミナントネガティブなＭｙＤ８８ｌｐｒを発現するＲＡＷ ２６４．７細胞では、ＣｐＧオリゴヌクレオチド処理、ＭＰＬまたはＬＰＳポジティブコントロール（図２Ａ）によって誘導されたκＢ依存性ルシフェラーゼ活性が阻害された。ドミナントネガティブなＭｙＤ８８によるルシフェラーゼ活性の阻害程度は、トランスフェクションにおいて用いられるＭｙＤ８８ｌｐｒプラスミドの量（図２Ａ）およびＭｙＤ８８ｌｐｒ発現の得られるレベルに依存した（データは示さず）。ＩＬ−１８またはＩＬ−１（これらのレセプターは、シグナル伝達のためにＭｙＤ８８を必要する）の誘導された発現によって媒介されるＣｐＧオリゴヌクレオチドの間接的な効果は、これらの結果を説明し得る。しかし、ＲＡＷ ２６４．７マクロファージを組換えＩＬ−１８で処理した場合に得られたネガティブな結果（上記を参照のこと）に加えて、活性なＩＬ−１８は、それぞれ、免疫ブロッティングまたはＥＬＩＳＡによってＲＡＷ２６４．７細胞の抽出物においても上清においても検出可能ではなかった（データは示さず）。
【００６２】
ＣｐＧオリゴヌクレオチドにおけるメチル化状態およびＣＧジヌクレオチド配列に対するκＢ−ルシフェラーゼの活性化およびＭｙＤ８８ｌｐｒの阻害の依存性を調べた。ＣＧ配列を置換するＧＣを有するコントロールのオリゴヌクレオチド（ＧＣオリゴ）またはＣではなくメチル−Ｃを用いて合成されたコントロールのオリゴヌクレオチド（メチルＣＧ）を、κＢ−Ｌｕｃ単独で、またはＭｙＤ８８ｌｐｒでトランスフェクトされたＲＡＷ ２６４．７細胞に添加した。図２Ｂに関して、ＲＡＷ ２６４．７マクロファージを、κＢ−Ｌｕｃ単独（黒棒）またはκＢ−Ｌｕｃ＋ＭｙＤ８８ｌｐｒ（白棒）（１：１のプラスミド比）でトランスフェクトした；総ＤＮＡ濃度を、空のベクタープラスミドの添加によって一定に保った。トランスフェクトした細胞を、非メチル化ＣＧモチーフを含むオリゴヌクレオチド（４μＭ）、非メチル化ＧＣ（ＧＣ）もしくはＣｐＧモチーフを置換するメチル化ＣＧモチーフ（ｍＣＧ）を有する類似のオリゴヌクレオチド、またはＣＧモチーフを欠く無関係の配列（非ＣｐＧ）で、翌日、６時間にわたって処理した。示した結果は、未刺激のトランスフェクトしたコントロールの平均値（「未処理」カラム）に対して正規化された、２連のウェルの平均＋／−標準偏差であり、そして２つの実験において得られた結果の代表である。図２Ｂに示されるように、ＣＧ配列のメチル化またはＧＣでのそれらの置換は、このオリゴヌクレオチドによるほぼ全てのκＢ−Ｌｕｃの活性化を無くした。ＣｐＧモチーフを欠くオリゴヌクレオチドで処理した細胞における基本的なルシフェラーゼ活性は、ＭｙＤ８８ｌｐｒ発現によって阻害されなかった。これらの結果は、ＣｐＧオリゴヌクレオチドによって活性化されたＭｙＤ８８依存性レセプターが、細菌ＤＮＡを模倣することが意図された非メチル化ＣＧ配列についての予測された特異性を有することを示唆する。
【００６３】
別のセットの実験では、シグナルを伝達する能力を欠く３つのＭｙＤ８８構築物をＰＣＲによって増幅し、そしてＣＭＶプロモーター（ｐＣＭＶ−ＦＬＡＧベクター、Ｓｉｇｍａ Ａｌｄｒｉｃｈ）の構成的制御下に置いた。第１の構築物ＭｙＤ８８ｌｐｒは、細胞死ドメインに点変異Ｆ５６Ｎを含む。第２の構築物ＭｙＤ８８−ΔＤＤは、細胞死ドメインを完全に欠く。第３の構築物ＭｙＤ８８−ＴＨＤは、細胞死ドメインおよび中間ドメインの両方を欠く。ＤＮＡを、カチオン性脂質試薬を用いてＲＡＷ ２６４．７細胞またはマウスの胚性ＮＩＨ−３Ｔ３細胞にトランスフェクトした。ルシフェラーゼレポータープラスミドを同時トランスフェクトした。上流のκＢ依存性プロモーターのプロセシング。ルシフェラーゼ転写は、ＮＦ−κＢの放出を当てにする。ＮＦ−κＢの放出は、Ｔｏｌｌシグナル伝達経路における最終的な事象の１つである。次いで、細胞を、ＣｐＧオリゴヌクレオチドおよびいくつかのコントロールのサイトカインで刺激した。ルシフェラーゼ発現を、ルミノメーターでアッセイして、変異ＭｙＤ８８タンパク質がＣｐＧシグナルに影響を与えたか否かを決定した。制限分析および配列決定の結果は、ＭｙＤ８８構築物の存在を確認した（データは示さず）。ドミナントネガティブなＭｙＤ８８はＣｐＧオリゴヌクレオチド刺激に応じてシグナル伝達を特異的かつ選択的にブロックする（図３Ａおよび３Ｂ）。この構築物は、ＴＮＦ ＭｙＤ８８依存性経路を経るシグナル伝達に影響を与えなかった（ネガティブコントロール）が、ＭｙＤ８８依存性ＬＰＳシグナル伝達およびＩＬ−１シグナル伝達を阻害した（図３Ａ）。ＭｙＤ８８ ＴＩＲドメイン単独は、シグナル伝達をブロックするために十分であった。
【００６４】
これらの結果は、ＭｙＤ８８機能が、ＣｐＧオリゴヌクレオチドおよびＭＰＬによるＮＦ−κＢの完全な活性化に必要とされることを実証する。ＭｙＤ８８を必要とする全ての公知のレセプターは、共通の構造的特徴ＴＨＤを共有するので、この知見はまた、推定のＣｐＧレセプターの構成要素がＴＨＤを有することを暗示した。ＴＨＤを有するレセプターの存在およびＣｐＧモチーフによって誘導されるシグナル伝達におけるＴｏｌｌ経路構成要素の関与は、新規である。
【００６５】
この結果は、ＣｐＧレセプター構成要素の同様の構造的特徴において第１の洞察を提供する。非メチル化ＣｐＧモチーフを含むオリゴヌクレオチドに対する免疫応答がＴＨＤを含むレセプターを通して媒介されることを知ることによって、対応する遺伝子を同定する新たなアプローチが提供されることが当業者によって認識される。ＣｐＧオリゴヌクレオチドのＭｙＤ８８依存性効果のメチル化依存性および良好な配列特異性、ならびにＡＰＣの他のヌクレオチドベースアクチベーターに対するこれらの知見の関連性の調査が行われ得る。
【００６６】
（実施例４：ＴＬＲ４はＣｐＧシグナル伝達に必要とされない）
ＣｐＧ−Ｒのポリペプチド配列を決定する好ましい方法は、公知のＴＬＲが有望な候補であり得るか否かを試験することである。マウスにおけるＬＰＳに対する応答についてのＴＬＲ４についての必要条件は、ＴＬＲ４に点変異を有するエンドトキシン非応答性系統Ｃ３Ｈ／ＨｅＪ、ならびにＴＬＲ４を発現しないＣ５７Ｂｌ／ｌ０ＳｃＣｒマウスおよびＣ５７Ｂｌ／ｌ０ＳｃＮＣｒマウスを用いて実証されている（Ｖｏｇｅｌら，Ｊ．Ｉｍｍｕｎｏｌ．，１９９９，１６２，５６６６−７０，Ｑｕｒｅｓｈｉら，Ｊ．Ｅｘｐ．Ｍｅｄ．，１９９９，１８９，６１５−２５，Ｃｈｏｗら，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，１９９９，２７４，１０６８９−９２，Ｈｏｓｈｉｎｏら，Ｊ．Ｉｍｍｕｎｏｌ．，１９９９，１６２，３７４９−５２およびＰｏｌｔｏｒａｋら，Ｓｃｉｅｎｃｅ，１９９８，２８２，２０８５−８）。
【００６７】
インビトロでのＣｐＧオリゴヌクレオチドおよびＭＰＬに対するＡＰＣの応答におけるＴＬＲ４の役割を評価するために、ＬＰＳ応答性マウス（Ｂａｌｂ／ｃ）およびＣ３Ｈ／ＨｅＪマウス由来のＢＭＤＤＣを、これらのアジュバントとともに一晩培養し、そしてＤＣ活性化／成熟のマーカーのアップレギュレーションをアッセイした。細胞の表面のＣＤ８６発現を、フローサイトメトリーによって定量した。ＣＤ８６は、活性化された樹状細胞およびマクロファージにおいてアップレギュレートされるＮＦ−κＢ標的遺伝子である。これは、抗原特異的Ｔ細胞を活性化する能力を増強する。図４に関して、野生型（Ｂａｌｂ／ｃ；黒棒）およびＴＬＲ４変異マウス（Ｃ３Ｈ／ＨｅＪ；白棒）由来のＢＭＤＤＣを、上記のように、ＧＭ−ＣＳＦ中で６日間にわたって増殖させ、次いで、ＣｐＧオリゴヌクレオチド（５μＭ）、コントロールオリゴヌクレオチド（５μＭ）もしくはＬＰＳ（１μｇ／ｍｌ）で一晩処理したか、または未刺激のままにした。生きたＣＤ１１ｃ陽性細胞上でのＣＤ８６の細胞表面発現を、フローサイトメトリーによってアッセイした。示した結果は、同じ培養物由来の未処理のＢＭＤＤＣの相乗平均蛍光（ＭＦ）に対して正規化した、アジュバントで処理したＢＭＤＤＣのＭＦであり、そして３つの実験において得られた結果の代表である。図４に示すように、ＣｐＧオリゴヌクレオチド、ＬＰＳで処理した野生型ＢＭＤＤＣは、細胞表面ＣＤ８６発現の増加を示した。ＴＬＲ４変異ＢＭＤＤＣでは、ＣＤ８６発現における変化はＭＰＬ処理細胞でもＬＰＳ処理細胞でも観察されず、一方、野生型ＢＭＤＤＣにおいて観察された増加と同様の、ＣＤ８６発現における増加が、ＣｐＧオリゴヌクレオチド処理培養物において見られた（図４およびデータは示さず）。従って、ＴＬＲ４は、ＬＰＳおよびＭＰＬによるインビトロでのＡＰＣ活性化に必要とされるが、ＣｐＧオリゴヌクレオチドによるインビトロでのＡＰＣ活性化には必要とされない。
【００６８】
上記の実験に類似したさらなる実験を実施して、ＣｐＧ−Ｒの正確な性質をより良く特徴付けるために他の公知のＴｏｌｌ様レセプターを試験し得る。これを、公知のインタクトなＴＬＲおよびこれらのレセプターを非機能的にする変異を有する任意の匹敵する細胞株において実施し得る。インタクトな場合にＣｐＧによって活性化されるがレセプターが欠損している場合は活性化されないレセプターを有する細胞の同定は、どのＴＬＲが存在するかおよびどの特定のＴＨＤが存在するかを正確に指摘する。
【００６９】
本明細書中に開示される結果はまた、シグナル伝達経路についてアダプタータンパク質ＭｙＤ８８を必要とするレセプターによって媒介される細胞のシグナル伝達のアゴニストまたはアンタゴニストとして作用し得る化合物を開発するために有用なアプローチを提供することが当業者によって認識される。化合物は、細胞とともにインキュベートされ得、そしてＣｐＧ活性化から続くことが公知の事象のカスケードがもたらされたかを見るためにアッセイされ得る。次いで、細胞は、ＭｙＤ８８ｌｐｒ遺伝子でトランスフェクトされ得る。ＭｙＤ８８ｌｐｒ遺伝子を伴わないコントロールにおいて効果を生じるが、ＭｙＤ８８ｌｐｒ遺伝子が発現した場合に効果を生じない化合物は、ＭｙＤ８８アダプタータンパク質を必要とする経路を経た細胞内シグナル伝達を活性化することが示されている。
【００７０】
（実施例５：ＣｐＧ−Ｒの単離）
多数の異なる手順を利用して、Ｔｏｌｌ関連のＣｐＧ−Ｒ構成要素を同定し得る。第１に、当業者は、公知でかつ新規のＴｏｌｌ様レセプターが、ＣｐＧオリゴヌクレオチドに対する応答性を非応答性細胞に付与する能力を調べ得る。第２に、当業者は、ＭｙＤ８８を使用して、ＣｐＧオリゴヌクレオチドによって特異的に活性化され、そして他のＴｏｌｌレセプターリガンド（例えば、ＬＰＳ）によっては活性化されない相互作用タンパク質を精製し得るか、または酵母ツーハイブリッドシステムを使用して、対応するｃＤＮＡを単離し得る。
【００７１】
マウスＴｏｌｌ様レセプター１〜６（ＴＬＲ１〜６）は、密接に関連したヒトホモログを有する。完全なまたは部分的な配列は、マウスの遺伝子についての公のデータベースにおいて利用可能である。ＴＬＲをコードする発現ベクターは、ＣｐＧオリゴヌクレオチド（例えば、κＢ−ルシフェラーゼ）によって活性化されるレポーター遺伝子とともに、ＣｐＧ非応答性細胞株（例えば、ＮＩＨ３Ｔ３）中にトランスフェクトされ得る。同時トランスフェクトされたＴＬＲのいずれかがＣｐＧオリゴヌクレオチドに対する応答性を付与する場合、これは、ＣｐＧオリゴヌクレオチドでの処理の際にレポーター遺伝子の発現によって反映される。
【００７２】
類似のアプローチを用いて、新規なＴＬＲを含むＣｐＧシグナル伝達における任意の候補ＣｐＧ−Ｒ構成要素の関与を試験し得る。新規のＴｏｌｌ関連レセプターを同定およびクローニングするためのアプローチとしては、ゲノミックス、配列データベースのスクリーニング、縮重ＰＣＲ、酵母ツーハイブリッドシステムおよびハイブリダイゼーションによるライブラリースクリーニングが挙げられる。活性なＣｐＧオリゴヌクレオチドは、霊長類ではなく、げっ歯類において最も良く記載されているので、マウスの系において研究して、Ｔｏｌｌ関連ＣｐＧ構成要素を同定し、次いで例えばＰＣＲを用いてヒトのホモログを単離することが好ましい。
【００７３】
ＭｙＤ８８ｌｐｒは、インタクトなＴｏｌｌ相同性ドメインおよび単一の不活化点変異を細胞死ドメイン内に含み、そして細菌または真核生物細胞のいずれかにおいて組換えタンパク質として発現され得る。組換えタンパク質をアフィニティー試薬として用いて、ＣｐＧオリゴヌクレオチド処理細胞（例えば、ＲＡＷ２６４．７）から相互作用タンパク質を単離および精製し得る。ＴＬＲシグナル伝達経路における下流のエフェクター分子（例えば、ＩＲＡＫ、ＴＲＡＦ６）の結合は、細胞死ドメインにおける「ｌｐｒ」変異によって最少になるかまたは除去されるべきである。従って、ＭｙＤ８８ｌｐｒに特異的に結合するタンパク質は、ＴＨＤおよび／またはＴＨＤを細胞死ドメインに連結する領域と相互作用するタンパク質である。Ｔｏｌｌ相同性ドメインは同質型相互作用を介して会合するので、ＴＨＤを含むＣｐＧ−Ｒ構成要素は、ＭｙＤ８８ ＴＨＤに結合することが予想される。一旦、推定のＣｐＧ−ＲがＭｙＤ８８についてアフィニティー精製されると、対応するｃＤＮＡは、例えば、ペプチド分析およびＰＣＲのような標準的な方法によって単離され得る。
【００７４】
同様に、ＭｙＤ８８ｌｐｒは、酵母ツーハイブリッドシステムにおいてベイトとして用いられて、相互作用タンパク質をコードするｃＤＮＡを単離し得る。次いで、ＣｐＧオリゴヌクレオチド誘導性シグナル伝達に対するこれらのｃＤＮＡの関連性が上記の通りに試験され得る。
【図面の簡単な説明】
【図１Ａ】 図１Ａは、ＲＡＷ ２６４．７マクロファージにおけるＮＦ−κＢの活性化を示す棒グラフである。ここでＲＡＷ ２６４．７細胞を、κＢで駆動したルシフェラーゼレポーター遺伝子でトランスフェクトし、そして２４時間後にインターロイキン−１（ＩＬ−１；２０ｎｇ／ｍｌ）、ＭＰＬ（１μｇ／ｍｌ）、ＬＰＳ（１μｇ／ｍｌ）またはホスホロチオエートＣｐＧオリゴヌクレオチド（４μＭ）で６時間処理した。
【図１Ｂ】 図１Ｂは、ＣｐＧオリゴヌクレオチド誘導性κＢ−ルシフェラーゼ（κＢ−Ｌｕｃ）活性化を示すグラフである。ここで、ＲＡＷ ２６４．７細胞を、κＢ−ルシフェラーゼレポータープラスミドで一過性にトランスフェクトし、そして２４時間後に示された濃度の、ＣｐＧモチーフを含むホスホロチオエートオリゴヌクレオチド（０．２５〜８μＭ）またはコントロールオリゴヌクレオチド（４μＭ）で６時間処理した。
【図１Ｃ】 図１Ｃは、ＣｐＧオリゴヌクレオチドまたはＬＰＳによるκＢ−ルシフェラーゼ活性化の反応速度論を示すグラフである。ここで、ＲＡＷ ２６４．７細胞を、κＢ−Ｌｕｃで一過性にトランスフェクトし、そして２４時間後に、ＣｐＧオリゴヌクレオチド（４μＭ；白丸）またはＬＰＳ（１μｇ／ｍｌ；黒丸）で処理した。
【図２Ａ】 図２Ａは、ドミナントネガティブ変異体ＭｙＤ８８（ＭｙＤ８８ｌｐｒ）が、ＣｐＧオリゴヌクレオチド誘導性κＢ−ルシフェラーゼ活性化を阻害することを示すグラフである。ここで、ＲＡＷ ２６４．７細胞を、κＢ−Ｌｕｃ（１μｇ）単独（黒棒）またはＭｙＤ８８ｌｐｒをコードするプラスミド（１０：１（灰色棒）もしくは１：１（白棒）の２つの比で）でトランスフェクトした。
【図２Ｂ】 図２Ｂは、ＮＦ−κＢの活性化およびＭｙＤ８８ｌｐｒによる阻害が、非メチル化ＣＧ配列に依存することを示す棒グラフである。ここで、ＲＡＷ ２６４．７マクロファージを、κＢ−Ｌｕｃ単独（黒棒）、またはκＢ−ＬｕｃおよびＭｙＤ８８ｌｐｒ（白棒）（１：１のプラスミド比）でトランスフェクトした。
【図３Ａ】 図３Ａは、ＭｙＤ８８ドミナントネガティブ構築物をコードするプラスミドでトランスフェクトしたＮＩＨ−３Ｔ３細胞の代表的なルシフェラーゼアッセイを示す棒グラフである。
【図３Ｂ】 図３Ｂは、ＭｙＤ８８ドミナントネガティブ構築物をコードするプラスミドでトランスフェクトしたＲＡＷ ２６４．７細胞の代表的なルシフェラーゼアッセイを示す棒グラフである。
【図４】 図４は、ＣｐＧオリゴヌクレオチド処理に対するＡＰＣ応答についてのＴｏｌｌ様レセプター４（ＴＬＲ４）非依存性を示す棒グラフである。ここで、野生型（Ｂａｌｂ／ｃ；黒棒）およびＴＬＲ４変異マウス（ＣＨ３／ＨｅＪ；白棒）に由来する未成熟骨髄由来樹状細胞（ＢＭＤＣＣ）を、上記のようにＧＭ−ＣＳＦ中で6日間増殖させ、次いで、ＣｐＧオリゴヌクレオチド（５μＭ）、コントロールオリゴヌクレオチド（５μＭ）、またはＬＰＳ（１μｇ／ｍｌ）で一晩処理したか、あるいは刺激しないままにした。
【配列表】

[0001]
(Citation of related application)
No. 60 / 163,157 filed on Nov. 2, 1999 and No. 60/167 filed on Nov. 24, 1999, under 35 USC 119 (e). , 389 claim priority. Each of these is hereby incorporated by reference in its entirety.
[0002]
(Field of Invention)
The present invention relates generally to the identification of immune responses and proteins, and pathways for signaling immune responses, specifically the CpG receptor (CpG-R).
[0003]
(Background of the Invention)
The mammal's unique immune system recognizes and responds to molecular features characteristic of pathogenic organisms. Different parts of pathogens (eg surface proteins, especially cell wall components and specific nucleotide sequences) can be recognized and can elicit different immune responses. It has long been known that cells have a variety of receptors and membrane-bound proteins that recognize foreign elements and elicit cascades known as immune responses. Two broad classes or types of responses are well known: humoral immunity or antibody-mediated immunity; and cell-mediated immunity. Certain pathogens or conditions can be effectively controlled primarily by antibody-mediated reactions, while other conditions or pathogens require a strong cellular response to mediate host defense.
[0004]
An adjuvant is a compound that can enhance the intrinsic immune response. Adjuvants can enhance both humoral and cellular immunity. For some conditions or diseases, such as those caused by human immunodeficiency virus or hepatitis C virus, it is particularly desirable to increase the intrinsic cell-mediated immune response by administration of an adjuvant.
[0005]
Unmethylated CG dinucleotide sequences (commonly found in bacterial DNA but not found in mammalian DNA) have been found to stimulate the unique immune system (Lipford et al., Trends Microbiol., 1998, 6, 496-500, Carson et al., J Exp. Med., 1997, 186, 1621-2, and Krieg et al., Trends Microbiol., 1998, 6, 23-7). Exposure to unmethylated CG oligonucleotides or motifs in natural DNA or synthetic oligonucleotides can be achieved by antigen-presenting cells (APC), dendritic cells and macrophages (Jakob et al., J. Immunol., 1998, 161, 3042-9, Sparwasser et al., Eur. J Immunol., 1998, 28, 2045-54, Sparwasser et al., Eur. J. Immunol., 1997, 27, 1671-9, Stacey et al., J. Immunol., 1996, 157, 2116-22. , And Jakob et al., Int. Archives Allergy Immunol., 1999, 118, 457-461), and B cells (Krieg et al., Nature, 1995, 374, 546-9) are directly active. To. NK cells and T cells are also activated by exposure to oligonucleotides containing CpG motifs, but some of these effects are indirectly mediated by cytokines produced by other cell types. (Bendigs et al., Eur. J. Immunol., 1999, 29, 1209-18, Sun et al., J. Exp. Med., 1998, 188, 2335-42, and Ballas et al., J. Immunol., 1996, 157. , 1840-5).
[0006]
In experimental vaccines, including an oligonucleotide containing a CpG motif as an adjuvant, either by direct incorporation into plasmid DNA or by co-administering a synthetic CpG oligonucleotide with a protein antigen, increased antibody titers (particularly , IgG2a subtype). Oligonucleotides containing CpG motifs have also been shown to be potent stimulators of cytolytic T cell activity, which is characteristic of cell-mediated immune responses (Brazolot Millan et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15553-8, Davis et al., J. Immunol. 1998, 160, 870-6, Davis Mt. Sinai J. Med., 1999, 66, 84-90, and McCluskie et al., J. Immunol., 1998. 161, 4463-6). The ability of oligonucleotides containing CpG motifs to induce the expression of Th1 cytokines (particularly interleukin-12 (IL-12) and interferon-γ (IFN-γ)) is that oligonucleotides containing CpG motifs have a variety of antigens ( Notably, it appears to be responsible for the strong Th1 bias of the immune response observed when used as an adjuvant for hepatitis B). Th1 cell mediated immunity is believed to be necessary for protection against many vaccine targets currently under investigation (eg, the aforementioned human immunodeficiency virus). Oligonucleotides containing CpG motifs have also been shown to confer protection against pathogens such as Listeria and Leishmania, even when administered without antigen associated with CpG oligonucleotide treatment (Zimmermann et al., J. Immunol., 1998, 160, 3627-30, Lipford et al., Eur. J. Immunol., 1997, 27, 3420-6, and Walker et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 6970-6975). It may have therapeutic applications in treating microbial infections. Furthermore, CpG oligonucleotide treatment protects immature B cells from apoptosis. Furthermore, CpG oligonucleotide activity is an unspecified result and is a specific methylation in that unmethylated CG with 5 ′ adjacent purines and 3 ′ adjacent pyrimidines has the greatest effect. Pretreatment with CpG oligonucleotides has a protective effect in allergic and LPS-induced asthma models (Schwartz, J. Immunol., 163, 224 and Sur et al., J. Immunol., 162, 6284), And in vitro IgE production by human peripheral blood monocytes from atopic patients can be reduced.
[0007]
The mechanism by which CpG oligonucleotides interact with cellular receptors and initiate intracellular signaling is unknown. DNA containing the CpG motif is initiated via the endocytotic pathway in the context of oligomeric or plasmid DNA and accumulates in endosomes (Hacker et al., EMBO J., 1998, 17, 6230-40, Yi et al., J. Immunol., 1998, 161, 4493-7, Yi et al., J Immunol., 1998, 160, 4755-61, and Macfarlane et al., J. Immunol., 1998, 160, 1122-31). This uptake may not be sequence specific because it can be competed by DNA lacking the CpG motif (Hacker et al., Supra). Responses to oligonucleotides containing CpG motifs are such that cellular uptake (Krieg et al., 1995, supra) and chloroquine and other blockers of endosomal maturation can inhibit cellular and molecular responses to oligonucleotides containing CpG motifs. Endosomal acidification may be required. The exact mechanism of this inhibition remains unresolved and the subcellular location of any CpG oligonucleotide related protein or putative CpG receptor also remains unresolved.
[0008]
The mechanism by which cells recognize oligonucleotides containing CpG motifs has not been described so far, but it is known that CpG oligonucleotides activate multiple intracellular signaling pathways in macrophages and B cells. . Specifically, stress-activated MAP kinases, JNK and p38, and their transcription factor targets AP-1 and ATF-2 are phosphorylated in response to CpG oligonucleotide treatment (Hacker et al., EMBO J. et al. 1998, 17, 6230-40, and Yi et al., J. Immunol., 1998, 161.4493-7). A pathway leading to nuclear translocation of NF-κB (which includes a kinase cascade leading to phosphorylation and degradation of inhibitory subunits), IκB (Zandi et al., Mol. Cell. Biol., 1999, 19, 4547-51). (See also Yi et al., J. Immunol., 1998, 160, 1240-5, and Yi et al., J. Immunol. 1998.160, 4755-61). ).
[0009]
Other immunostimulatory agents are known to have similar effects. Monophosphoryl lipid A (MPL) induces a Th1 lymphocyte response (Ullrich et al., Monophosphoryl Lipid A as an Adjuvant in Vaccine Design: The Subunit and Adjuvant. Is known to those skilled in the art. In macrophages, the MAPK and NF-κB pathways are activated by other drugs, notably bacterial endotoxin, lipopolysaccharide (LPS). Nuclear NF-κB (often in combination with other transcription factors such as AP-1) is required for induction of transcription of many immune response genes (costimulatory molecules and cytokines) (Baldwin, Annu). Rev. Immunol., 1996, 14, 649-83).
[0010]
In mice, genetic evidence relates to immune responsiveness to bacterial endotoxins against Toll-like receptor (TLR) 4, a Toll / IL-1 receptor family. Experiments with human TLRs relate to TLR2 and / or TLR4 in the LPS response (reviewed in Qureshi et al., Trends in Genetics, 1999, 15, 291-294). More recently, additional bacterial products (including peptidoglycan and mycobacterial lipoprotein) have also been shown to act through members of the Toll receptor family (Yoshimura et al., J. Immunol., 1999, 163, 1). -5, Brightbill et al., Science, 1999, 285, 732-736, Aliplantis et al., Science, 1999, 285, 736-739, and Hirschfeld et al., J. Immunol., 1999, 163, 2382-2386). Toll-related receptors are conserved through millions of years of evolution; in insects and even in plants, Toll-related receptors are genetically responsive to various pathogens (bacteria, fungi, and at least one virus) (Reviewed in Hoffmann et al., Science, 1999, 284, 1313-1318). It is unknown whether oligonucleotides containing CpG motifs also act through Toll-related receptors or whether putative CpG receptors can contain Toll homology domains (THD).
[0011]
Due to the potential usefulness of these CpG oligonucleotides as stimulators of various immune responses, there has been considerable interest in the isolation and characterization of CpG-R and the mechanism of action and signaling pathways of CpG-R. Yes. Thus, the object of the present invention is to characterize and identify CpG-R, to distinguish other proteins or receptors that can lead to similar immune responses from CpG-R, and to have more desirable pharmacological properties, It is to develop a useful method that allows the design of novel CpG-R ligands that can retain the immunostimulatory properties of oligonucleotides containing CpG motifs.
[0012]
(Summary of the Invention)
The present invention relates to an isolated nucleic acid molecule encoding a portion, CpG-R or a fragment thereof, a nucleotide sequence complementary to at least a portion of a nucleotide sequence encoding CpG-R, and CpG-R or a fragment thereof. It relates to a nucleotide sequence homologous to the encoding nucleotide sequence.
[0013]
The invention also relates to a recombinant expression vector comprising any of the nucleic acid molecules described above and a host cell transformed with this vector.
[0014]
The invention also relates to a polypeptide encoding CpG-R, or a complex of polypeptides, or a homologue or fragment thereof. Such a polypeptide can be produced by introducing a recombinant expression vector containing any of the nucleic acid molecules described above into a compatible host cell and allowing the host cell to grow under conditions that allow expression of the polypeptide. And by isolating the polypeptide from the host cell.
[0015]
The invention also relates to a composition comprising any of the nucleic acid molecules or polypeptides described above and an acceptable carrier or diluent.
[0016]
The invention also relates to an isolated antibody that binds to an epitope on a polypeptide encoded by any of the nucleic acid molecules described above.
[0017]
The invention also relates to a kit comprising an antibody that binds to a polypeptide encoded by any of the nucleic acid molecules described above, and a control antibody.
[0018]
The invention also relates to a method of modulating a mammal's immune response by administering to the mammal an amount of a compound that binds to CpG-R or activates or inhibits CpG activation of CpG-R. .
[0019]
The invention also includes contacting a compound with a cell that expresses CpG-R or CpG-R, and whether the compound binds to CpG-R or whether the compound modulates the activity of CpG-R. It relates to a method for identifying compounds that bind to CpG-R or modulate the activity of CpG-R by determining whether or not.
[0020]
(Description of Preferred Embodiment)
The present invention is based on the surprising discovery of the identity of a specific CpG receptor (CpG-R). This is also further based on the surprising discovery that CpG-R or complexes containing it contain THD. The discovery of the presence of THD within this receptor made it possible to test for the need for known Toll receptors for CpG responses and further characterization of CpG-R. The present invention relates in particular to CpG-R, which can be a single polypeptide, or a complex of multiple polypeptides, and optionally further components such as polysaccharides, lipids, etc. Can be included. This CpG-R or complex comprising it preferably comprises THD and interacts with MyD88 adapter protein. In addition, CpG-R can bind to CpG oligonucleotides.
[0021]
The practice of the present invention uses conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the purview of those skilled in the art unless otherwise indicated. Such techniques are explained fully in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); DNA Cloning: A Practical Approach, Volumes 1 and 2 (D. Glover Ver.); Methods In Enzymolology (Methods In Enzymolology). Kaplan ed., Academic Press, Inc.); Fundamental Virology, 2nd edition, Volume 1 and 2 (BN Fields and DM Knipe edition), Remington's Pharmaceutical Sciences, 18th edition. , Pennsylvania: Mack Publishing Company, 1990); Handbook of Experimental Immunology, Volumes 1 to 4 (DM Weir and CC Blackwell, 1986, Blackwell Scientific Publics, and Handbook of Surface and Colloid. CR, 97). See Seymour / Carrahers Polymer Chemistrv (4th edition, Marcel Dekker Inc., 1996).
[0022]
As used herein, the term “CpG-R” refers to a CpG receptor, which can be a single polypeptide or a complex of multiple polypeptides, and optionally, For example, additional components such as polysaccharides, lipids and the like may be included. CpG-R can be a protein involved in the CpG signaling cascade or can be a receptor for binding CpG oligonucleotides.
[0023]
As used herein, the term “activity” refers to any activity or cascade of activities associated with binding or signaling (eg, as described above) of a CpG oligonucleotide.
[0024]
As used herein, the term “antibody” includes, but is not limited to, intact antibodies, their Fab fragments and F (ab) ₂ It is meant to refer to fragments, and chimeric antibodies.
[0025]
As used herein, the phrase “oligonucleotide comprising at least one CpG motif” or “CpG oligonucleotide” refers to a polynucleotide comprising at least one unmethylated CG dinucleotide sequence, preferably Refers to an oligonucleotide. An oligonucleotide comprising at least one CpG motif can comprise multiple CpG motifs.
[0026]
As used herein, the phrase “CpG motif” refers to an unmethylated dinucleotide portion of an oligonucleotide comprising a cytosine nucleotide followed by a guanosine nucleotide. 5-methylcytosine can also be used in place of cytosine.
[0027]
As used herein, the term “homologous” refers to the entire nucleotide or amino acid sequence encoding CpG-R, or at least a portion of CpG-R, at least about 70%, more preferably, Refers to a nucleotide or amino acid sequence characterized by at least about 80%, more preferably at least about 90%, and most preferably at least about 95% sequence identity. Homologous amino acid sequences can include amino acid sequences that encode conservative amino acid substitutions. Sequence identity can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Mad.) Using default settings. This program uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is hereby incorporated by reference in its entirety).
[0028]
As used herein, the term “about” means that the value varies by ± 10% of that value.
[0029]
As used herein, the term “modulate” means an increase or decrease in the amount or effect of a particular activity or protein.
[0030]
One aspect of the invention relates to novel nucleic acid molecules comprising a novel nucleotide sequence encoding CpG-R. The nucleic acid molecule is preferably either RNA or DNA, but may include both RNA and DNA monomers or peptide nucleic acid monomers. Nucleic acid molecules can be single-stranded or double-stranded. Nucleic acid molecule monomers can be linked via conventional phosphodiester bonds or modified bonds, such as phosphorothioate bonds. Furthermore, the sugar moiety of the monomer can be modified, for example, by the addition of 2 ′ substituents, which confer nuclease resistance and / or assist in cellular uptake. The nucleic acid molecule can also include a nucleotide sequence that is complementary to at least a portion of the nucleotide sequence encoding CpG-R. Preferably, the nucleic acid molecule comprises a nucleotide sequence that is complementary to the entire sequence, but may comprise a nucleotide sequence that is complementary to a portion of the entire sequence. The nucleic acid molecule can also include a nucleotide sequence that is homologous to a nucleotide sequence encoding CpG-R, and relative to the entire sequence encoding CpG-R or any portion thereof, as determined above. , At least about 70% homology, more preferably at least about 80% homology, more preferably at least about 90% homology, and most preferably at least about 95% homology.
[0031]
A wide variety of alternative cloning and in vitro amplification methods are well known to those of skill in the art. Examples of these techniques are described, for example, by Berger et al., Guide to Molecular Cloning Technologies, Methods in Enzymology 152 Academic Press, Inc. San Diego, CA (Berger), which is hereby incorporated by reference in its entirety.
[0032]
Another aspect of the invention relates to a vector or recombinant expression vector comprising any of the nucleic acid molecules described above. A vector is used herein to amplify either CpG-R encoding DNA or RNA in order to express DNA encoding CpG-R. Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments. Preferred viral particles include, but are not limited to, adenovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus and retrovirus. Preferred expression vectors include pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech), pGEM vector (Promega), pPROEX vector (LTI, Bethesda, MD), Bluescript vector (Stratagene), pQE vector (SEiagen, it, 420) And pYES2 (Invitrogen), but is not limited thereto.
[0033]
A preferred expression vector is a replicable DNA construct in which a DNA sequence encoding CpG-R is operably linked to appropriate control sequences, which control sequences are expressed in the appropriate host or organism by the CpG-R. Can lead to the expression of DNA regions are operably linked when the regions are functionally arranged with respect to each other. Control sequences include, but are not limited to, promoters, operators, ribosome binding sequences, and transcription / translation termination sequences.
[0034]
Preferred vectors preferably contain a promoter that is recognized by the host organism. The promoter sequences of the present invention can be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the bacteriophage λ P _R Promoter and P _L Promoters (The Bacteriophage Lambda, Hershey, Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), Lambda II, Hendrix, R. W. S (1980), each of which is incorporated herein by reference in its entirety); E. coli trp promoter, recA promoter, heat shock promoter and lacZ promoter, and SV40 early promoter (Benoist et al., Nature, 1981, 290, 304-310, which is hereby incorporated by reference in its entirety) ). Additional promoters include mouse mammary carcinoma virus, human immunodeficiency virus long terminal repeat, maloney virus, cytomegalovirus early promoter, Epstein-Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human Examples include, but are not limited to, muscle creatine and human metallothionein. In addition, suitable expression vectors can include markers that allow screening of transformed host cells. Expression vectors can be prepared by standard methods.
[0035]
Another aspect of the present invention relates to a transformed host cell having an expression vector comprising any of the nucleic acid molecules described above. Suitable host cells for expression of the polypeptides of the present invention include, but are not limited to, prokaryotes, yeast and eukaryotes. Suitable prokaryotic cells include, but are not limited to, bacteria of the genus Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus. Suitable eukaryotic cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), mouse 3T3 fibroblasts. Suitable yeast cells include, but are not limited to, the genus Saccharomyces, Pichia and Kluberomyces. The polypeptides of the present invention can also be expressed using a baculovirus expression system (Luckow et al., Bio / Technology, 1988, 6, 47, Baculovirus Expression Vector: A Laboratory Manual, O'Rilly et al., Ed., W H. Freeman and Company, New York, 1992 and US Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety. Furthermore, the MAXBACJ complete baculovirus expression system (Invitrogen) can be used, for example, for production in insect cells. The growth of such cells in cell culture is described, for example, in Tissue Culture, Academic Press, Kruse and Patterson (1973), which is hereby incorporated by reference in its entirety. Such a conventional procedure.
[0036]
In another aspect of the invention, it relates to an isolated polypeptide encoded by the nucleic acid molecule described above. In a preferred embodiment of the invention, the isolated polypeptide comprises an amino acid sequence encoding CpG-R. Alternatively, the polypeptide is a fragment of a polypeptide encoding CpG-R. Alternatively, the polypeptide comprises an amino acid sequence that is homologous to CpG-R or a fragment thereof. At least about 70% sequence identity or homology (as determined above), at least about 80% sequence identity or homology to CpG-R, preferably about 90% sequence identity Polypeptides having amino acid sequences having sex or homology, more preferably about 95% sequence identity or homology, and most preferably about 98% sequence identity or homology are encompassed by the present invention. Is intended. Preferred homologous polypeptides contain at least one conservative amino acid substitution compared to native CpG-R. Other preferred homologous polypeptides are up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acids compared to native CpG-R. Includes substitution. These polypeptides can be expressed in host cells as fusion proteins that can include regions from heterologous proteins. The polypeptides of the present invention can also include regions that are the same protein but differ in sequence from naturally occurring polypeptides. Furthermore, a homologous CpG-R peptide has at least about 70% functional homology, at least about 80% functional homology, preferably about 90% functional homology compared to CpG-R, More preferably, it comprises a polypeptide having about 95% functional homology, and most preferably about 98% functional homology. Accordingly, the present invention encompasses proteins that are homologous to CpG-R and essentially have at least one biological property (functional homology) that is substantially similar to that of CpG-R. .
[0037]
Polypeptides of the invention encoding CpG-R may be used for the ability to bind CpG oligonucleotides, for including a THD domain, and for the ability to interact with MyD88 adapter protein, eg, a recombinant expression library or sequence database Can be isolated by screening and the like. The MyD88 adapter domain is required for signal transduction from receptors for products such as polysaccharides and lipoproteins and interleukin-1. The polypeptides of the present invention are preferably provided in an isolated form, preferably substantially purified, and most preferably purified to homogeneity. The host cell is preferably lysed and the polypeptide is recovered from the lysate of the host cell. Alternatively, the polypeptide is recovered by purifying the cell culture medium from the host cells, preferably without lysing the host cells. Polypeptides can be recovered and purified from recombinant cell culture by well-known methods, including ammonium sulfate precipitation or ethanol precipitation, including anion exchange chromatography or cation exchange chromatography, hydroxyapatite chromatography, and lectin chromatography.
[0038]
Another aspect of the invention relates to compositions (including pharmaceutical compositions) comprising any of the nucleic acid molecules or polypeptides described above and an acceptable carrier or diluent. Preferably the carrier or diluent is pharmaceutically acceptable. The carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or in admixture with a wax. The formulations may also contain wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, thickening agents or flavoring agents. The formulations of the present invention may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by using means well known in the art. The pharmaceutical composition is sterilized and, if desired, mixed with adjuvants, emulsifiers, salts that affect osmotic pressure, buffers and / or colorants, etc. that do not deleteriously react with the active compound. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders can also be added.
[0039]
The polypeptides of the invention can be used to generate antibodies to the polypeptides of the invention and can be used to screen for compounds that modulate the activity of CpG-R or CpG oligonucleotides. Preferably, the antibody binds to an epitope within CpG-R. The antibody can be monoclonal or polyclonal. Hybridomas that produce antibodies that bind to the polypeptides of the invention and the antibodies themselves are useful for isolation and purification of the polypeptides. Furthermore, the antibody can be a specific inhibitor of CpG-R activity. Antibodies that specifically bind to a polypeptide of the invention can be used to purify the protein from natural sources or through recombinant techniques using well-known techniques and readily available starting materials. Methods for making antibodies are known to those skilled in the art. For techniques for preparing monoclonal antibodies, see, for example, Stites et al. (Ed.) Basic and Clinical Immunology (4th edition), Lange Medical Publications, Los Altos, CA (which is incorporated herein by reference in its entirety). See incorporated). Antibodies, Fab fragments and F (ab) ₂ Fragment production is described, for example, in Harlow, E .; And D. Lane (1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (Which is incorporated herein by reference).
[0040]
The present invention also relates to kits (including pharmaceutical kits). The kit can include any of the nucleic acid molecules, polypeptides or antibodies described above, and appropriate controls. The kit preferably comprises further components (eg instructions, solid support, reagents to aid quantification, etc.).
[0041]
Another aspect of the invention relates to a method of modulating an immune response in a mammal by administering to the mammal an amount of CpG-R, an antibody to CpG-R, or a compound that binds to CpG-R. The amount of CpG-R, an antibody to CpG-R, or a compound that binds to CpG-R depends on the animal species, animal size, etc., but can be determined by one skilled in the art. The route of administration can be any route that efficiently transports the active compound to the appropriate site of action or the desired site (eg, oral, nasal, rectal, pulmonary, transdermal or non-routed). Oral route, subcutaneous route, intravenous route, intraurethral route, intramuscular route, nasal route, ophthalmic solution, or ointment, parenteral or oral route are preferred).
[0042]
Another aspect of the invention relates to a method for identifying a compound that binds to either a nucleic acid molecule or polypeptide encoding CpG-R. The method includes contacting CpG-R or a nucleic acid molecule encoding the same with a compound and determining whether the compound binds to CpG-R or a nucleic acid molecule encoding the same. . Binding can be determined by binding assays well known to those skilled in the art. Examples of this assay include, but are not limited to, gel shift assay, Western blot, radiolabel competitive assay, simultaneous fractionation by chromatography, coprecipitation, ELISA, and the like. These assays are described in Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is hereby incorporated by reference in its entirety. The CpG-R polypeptide or nucleic acid molecule used in such a test is free in solution, bound to a solid support, bound to the cell surface, or localized within the cell. Can be either.
[0043]
Another aspect of the invention relates to a method of identifying a compound that modulates CpG-R signaling activity. The method includes contacting CpG-R with a compound and determining whether the compound modulates the activity of CpG-R. The activity in the presence of the test compound is measured and compared to the activity in the absence of the test compound. If the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound has increased activity. If the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound is inhibited in activity. The CpG-R used in such tests can be either free in solution in the presence of an appropriate substrate, bound to the cell surface, or localized within the cell. .
[0044]
Compounds that bind to and / or modulate CpG-R include, for example, vaccine adjuvants that promote cell-mediated immune responses, antibacterial agents (eg, protection from Listeria infection), tumor immunotherapy, allergy treatment (Eg, suppresses IgE in human PBMC and shifts from Th2 to Th1) and as an anti-inflammatory agent (eg, cystic fibrosis, sepsis, heart disease, chlamydia, inflammatory bowel disease, asthma, and multiple For use in sclerosis).
[0045]
The invention is further illustrated by the following examples, which are intended to clarify the invention. The foregoing examples are intended to illustrate the invention but are not to be construed as limiting the invention in any way. Those skilled in the art will recognize modifications that are within the spirit and scope of the invention. All references (including patents, applications, and printed publications) mentioned in this invention are intended to be incorporated herein by reference in their entirety.
[0046]
(Example)
(Example 1: General methodology)
(Animals and cell lines)
Bone marrow was collected from 6-12 week old female mice of various genotypes (C57B1 / 6, Balb / c; Charles River; or C3H / Hel, Jackson Labs). Fresh bone marrow cells were used or frozen in fetal calf serum (FCS; Summit) containing 10% dimethyl sulfoxide and stored at −80 ° C. Bone marrow macrophages (BMMO) were prepared as described in Current Protocols in Immunology, supra. In short, fresh or frozen bone marrow cells were treated with RPMI (10% heat inactivated (56 ° C., 30 minutes) fetal calf serum (FCS), 2 mM L-glutamine, 100 μg / ml streptomycin, 100 units / ml penicillin, 50 μM β-mercaptoethanol. And 100 U / ml recombinant macrophage colony stimulating factor (M-CSF; R & D Systems). After 24 hours, non-adherent cells were collected in a new petri dish and cultured for 7 days to produce a macrophage monolayer. Non-adherent BMDDCs were generated by culturing in the same medium supplemented with GM-CSF (200 U / ml; Preprotech) but not M-CSF. The RAW 264.7 mouse macrophage cell line (originally derived from Balb / c mice) was obtained from the American Type Culture Collection (ATCC). RAW 264.7 cells are cultured in DMEM with 10% heat-inactivated FCS, L-glutamine, streptomycin, penicillin, and 1 μM sodium pyruvate.
[0047]
(reagent)
Cell cultures were treated as indicated with the following reagents: K235 E.E. Coli LPS, purified gel filtration (Sigma), S. coli. Monophosphoryl lipid A (MPL; RIBI Immunochem Research, Inc) from minnesota R595 (sonicated for 10 minutes before addition). Oligos Etc (Wilsonville, OR); CpG oligonucleotide sequence, tccatgacgtttcctgacgtt (SEQ ID NO: 1); control non-CpG oligonucleotide tccaggacttctctcaggtt (SEQ ID NO: 2); oligonucleotide with phosphorothioate backbone was synthesized. In the same experiment, recombinant mouse interleukin-1β (1L-1) (Endogen) and interleukin-18 (IL-18) (Biosource) were also used to stimulate RAW264.7 cells. Additional CpG oligonucleotides that can be used in the present invention include, but are not limited to, oligonucleotides comprising, for example, the following nucleotide sequences:
[0048]
[Chemical]
(Under p14)

[0049]
.
[0050]
(Immunoblotting)
After stimulation, complete J protease inhibitor cocktail (Boehringer Mannheim) (Triton lysis buffer) in 1% Triton X-100, 50 mM Tris, 62.5 mM EDTA (pH 8.0 MO), pH 8.0 MO RAW 264.7 cells were lysed. Lysates were boiled in reducing sample buffer and separated on a 10% polyacrylamide gel using the NuPAGEJ Bis-Tris electrophoresis system (Novex). According to the manufacturer's instructions, the nitrocellulose membrane was probed with an antibody against IκB-α, phosphorylated-IκB-α (New England BioLabs), or anti-IL-18 (Santa Cruz Biotechnology) to enhance chemiluminescence ( Amersham) and visualized.
[0051]
(ΚB-Luc assay)
3 x 10 per well 24 hours prior to transfection ^Five At the cell density, RAW 264.7 cells were seeded in 6-well plates (Corning). The plasmids used in the transfection were pNF-κB-Luc (Clontech) and pCR3. V64-Met-Flag-MyD88lpr (donated by Jurgen Tschopp; described in Burns et al., J. Biol. Chem. 1998, 273, 12203-9). Total plasmid concentration was normalized across all wells by the addition of empty vector (pCMVKm2, Chiron). Cells were transfected with the indicated concentration of DNA-containing Opti-MEM I (Gibco BRL) with 10 ml of LipofectAMINE (Gibco BRL) per well according to the manufacturer's instructions. The cells were incubated with the transfection mixture for 3 hours at 37 ° C., then the culture medium was replaced and the cells were collected overnight. The next day, at the indicated concentrations and times, using phosphorothioate oligonucleotides, MPL, LPS or cytokines at 37 ° C., 5% CO ₂ The treated cells were treated in culture medium. Cells were washed once with cold phosphate buffered saline (PBS) and lysed using Reporter Lysis Buffer J (Promega). All luciferase activities in the lysis supernatant were determined using Microlite 2 plates (Dynex) and ML 3000 Luminometer (Dynatech) according to the manufacturer's instructions. For detection of FLAG-MyD88lpr expression, soluble pellets from transfected RAW 264.7 cells are resuspended in Triton lysis buffer and sonicated for 10 minutes, after which sample buffer is added. And boiled. After separation and transfer to a nitrocellulose membrane, the membrane was immunoblotted using FLAG M2 (Sigma) antibody.
[0052]
(Flow cytometry)
BMDDC were treated overnight with adjuvant as indicated, washed, and FcBlock (0.25 μg / 10 ⁶ Cells; Pharmingen), resuspended in cold PBS / 2% FBS, and FITC-conjugated and PE-conjugated antibodies (1 μg / 10 ⁶ Cells) was added after 5 minutes. Cells were incubated with antibodies on ice for 30 minutes, washed and analyzed by flow cytometry on a FACScan (Becton-Dickenson). This analysis included only cells from BMDDC cultures that stained positive for CD11c and had forward and side scatter characteristics of viable cells. Changes in CD86 expression are assessed based on the geometric mean fluorescence of CD86-FITC staining of viable CD11c cells treated with adjuvant normalizing the geometric mean CD86-FITC fluorescence of untreated BMDDC cultures.
[0053]
Example 2: CpG oligonucleotide activates MAPK pathway and NF-κB
Several biochemical responses of mouse BMMO and dendritic cells and mouse macrophage cell line RAW 264.7 to oligonucleotides containing or lacking the CpG motif were compared. As a model for the effect of the CpG motif on oligonucleotides, an oligonucleotide called 1826 (Chu et al., J. Exp. Med., 1997, 186, 1623-31) (in mice, its activity has been extensively reported in the literature. (Bachmeier et al., Science, 1999, 183, 1335-9). CpG and control oligonucleotides were synthesized into 20-mers each using a phosphorothioate backbone that enhances DNA stability (Agrawal et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 7595-9).
[0054]
As described in Example 1, macrophages were differentiated from bone marrow cells of C57B1 / 6 mice and then transferred to low serum medium lacking M-CSF for 4 hours. Lysates were prepared from cells treated with medium alone, phosphorothioate modified CpG oligonucleotide (1 μM), control oligonucleotide lacking CG sequence (1 μM), or MPL (100 ng / ml) for 30 minutes to recognize phosphorylated ERK1 Immunoblotting is performed with the antibody to be used and ERK. 2RAW 264.7 macrophages were similarly treated for 15 minutes with further treatment with LPS (100 ng / ml) and immunoblotted for phosphorylated ERK1 and ERK2. Activation of multiple mitogen activated protein kinase (MAPK) pathways (including ERK1 / 2, JNK and p38) by CpG oligonucleotides by immunoblotting of whole cell extracts from bone marrow from APC or RAW 264.7 cells Has been demonstrated. In all three cell types, phosphorylation of ERK, JNK and / or JNK substrate c-jun and phosphorylation of p38 MAPK substrate ATF 2 was observed within 1 hour of stimulation with CpG oligonucleotide, but the CpG sequence Were not observed in controls lacking (data not shown). These results indicate rapid activation of the JNK and p38 pathways by CpG oligonucleotides. The positive results for ERK1 / 2 MAPK activation in RAW 264.7 cells and BMMO are in contrast to observations in another mouse macrophage cell line J774, whereas ERK phosphorylation is observed in CpG oligonucleotide treated cells. Was not detected.
[0055]
The status of the NF-κB pathway in BMMO and RAW 264.7 cells treated with the panel adjuvant, including CpG oligonucleotide and control oligonucleotide, was also tested. BMMO was treated for 1.5 hours with LPS (1 μg / ml), MPL (1 μg / ml) phosphorothioate modified CpG oligonucleotide (4 μM) or control phosphorothioate oligonucleotide (non-CpG; 4 μM). Whole cell lysates were prepared and immunoblotted for IκBα as described above. In BMMO treated with LPS, MPL or CpG oligonucleotides, degradation of NF-κB inhibitory protein IκBα was observed within 1.5 hours, but not with control oligonucleotides (data not shown).
[0056]
Also lysed from cells treated with untreated RAW 264.7 cells or phosphorothioate CpG oligonucleotides (4 μM) for 5 minutes, 10 minutes, 20 minutes and 60 minutes and 60 minutes with control oligonucleotides lacking the CpG motif. A product was prepared. The lysates were then separated by electrophoresis and immunoblotted with antisera against phosphorylated IκBα and total IκBα. IκBα phosphorylation and degradation in RAW 264.7 cells showed similar specific activation by CpG oligonucleotides. This activation was detected at the level of IκBα phosphorylation within 10 minutes of CpG oligonucleotide addition, and IκBα degradation was significant at 20 minutes. Although IκBα was resynthesized in CpG oligonucleotide-treated RAW 264.7 cells within 1 hour, most IκBα was phosphorylated, suggesting continuous targeting for rapid degradation (data shown). )
[0057]
To directly assess κB-dependent transcription induced by CpG oligonucleotide or MPL treatment, an NF-κB-inducible reporter plasmid encoding luciferase under the control of multiple copies of the κ light chain enhancer (κB-Luc) RAW264.7 macrophages were transfected and treated the next day with adjuvant or cytokines. Specifically, RAW 264.7 cells were transfected with a kappa B driven luciferase reporter gene, and 24 hours later, IL-1 (20 ng / ml), MPL (1 μg / ml), LPS (1 μg / ml) Alternatively, it was treated with phosphorothioate CpG oligonucleotide (4 μM) for 6 hours. Luciferase activity in the cell lysate was quantified by luminometer and this result was normalized to the luciferase signal in untreated transfected cells. As shown in FIG. 1A, addition of CpG oligonucleotides to RAW 264.7 cells induced κB-dependent luciferase expression to levels similar to those induced by MPL or LPS. Activation of the κB reporter gene by CpG oligonucleotides was slightly weaker than that seen in response to MPL or LPS, both of which are strong activators of NF-κB in macrophages. IL-1 was a weak activator of κB-Luc in RAW 264.7 cells (FIG. 1A), as did IL-18 (data not shown).
[0058]
The effect of CpG oligonucleotides on κB-dependent luciferase expression was dose-dependent and time-dependent (see FIGS. 1B and 1C). Referring to FIG. 1B, RAW 264.7 cells transiently transfected with a kappa B-luciferase reporter plasmid were grown at concentrations of phosphorothioate CpG oligonucleotide concentrations ranging from 0.25 μM to 8 μM, or with control oligonucleotide (4 μM). Treated for 6 hours after 24 hours. Activation was detected at CpG oligonucleotide concentrations as low as 0.25 μM (FIG. 1B), but not at the lowest concentration tested (0.05 μM) (data not shown). The data shown in FIG. 1B is the average luciferase activity in lysates from duplicate wells normalized to luciferase levels in lysates from untreated transfected wells on the same 6-well tissue culture plate. . Reporter gene activation by CpG oligonucleotide treatment was maximal at 8 μM (FIG. 1B) and was not further enhanced at a dose of 10 μM (data not shown). Referring to FIG. 1C, RAW 264.7 cells transiently transfected with κB-Luc and treated 24 hours later with CpG-containing oligonucleotides (4 μM; open circles) or LPS (1 μg / ml; filled circles). The results shown in FIG. 1C are averages of duplicate wells normalized to untreated transfected wells. Exposure of RAW 264.7 cells transfected with κB-Luc to similar concentrations of control oligonucleotides lacking the CpG motif did not induce increased luciferase expression (FIGS. 1B and 1C). ΚB-dependent luciferase expression in RAW 264.7 macrophages showed similar kinetics in cells treated with CpG oligonucleotides or LPS (FIG. 1C); both treatments resulted in a rapid response (2-fold over 2 hours) This response reached a plateau in 4-6 hours.
[0059]
These results demonstrate that activation of the NF-κB pathway by CpG oligonucleotides in RAW 264.7 cells is specific, rapid, and dose dependent. The indirect effects of CpG oligonucleotides mediated by cytokines are unlikely given that the kinetics of signal transduction are very rapid. Addition of supernatant from CpG oligonucleotide treated RAW 264.7 cells to κB-Luc transfected RAW 264.7 macrophages induced little luciferase expression (less than 2 fold at 4 hours), It was confirmed that the CpG motif on κB-Luc transcription has a direct effect.
[0060]
(Example 3: Expression of dominant negative MyD88 blocks CpG induction of κB-dependent reporter gene)
Given the role of Toll receptors in response to various bacterial-derived structures (including LPS, peptidoglycans and lipoproteins), Toll-related receptors are thought to be involved in signal transduction in response to oligonucleotides containing unmethylated CpG sequences. It is done. Unmethylated CpG sequences are also pathogenic non-self features. To this end, RAW 264.7 cells are expressed in an expression vector encoding a kappa B-luciferase plasmid and a dominant negative form of MyD88, an adapter protein required for signaling by members of the Toll / IL-1 receptor family. Simultaneously transfected. The dominant negative MyD88 MyD88lpr used in these experiments has an intact Toll homology domain, but contains a point mutation in the death domain (DD). Similar mutations in the Fas cell death domain in lpr mice probably abolish Fas signaling by altering the conformation of this cell death domain, thus blocking the assembly of downstream signaling molecules. Overexpression of MyD88lpr in RAW 264.7 cells is expected to disrupt MyD88-dependent signaling by competing with endogenous MyD88 for association with proteins containing Toll homology domains. This effect of MyD88lpr is directed against Toll related proteins since mutations in this death domain must prevent association with downstream signaling components that may be shared with other activators of NF-κB. It is specific.
[0061]
Transfection of RAW 264.7 cells with a plasmid encoding FLAG-tagged MyD88lpr resulted in the expression of a protein of the expected size as determined by immunoblotting with anti-FLAG antibody (data not shown) . Referring to FIG. 2A, RAW 264.7 cells were then transformed into κB-Luc (1 μg) alone (black bars) or plasmid encoding MyD88lpr in two different ratios (10: 1 (gray bars) or 1: 1. (White bar)). Total plasmid DNA concentration was normalized across all wells by the addition of empty vector. Cells 24 hours after transfection were treated with CpG oligonucleotide (4 μM), control oligonucleotide lacking the CpG motif (4 μM), MPL (1 μg / ml) or LPS (1 μg / ml). Results shown are the mean +/- standard deviation of duplicate wells normalized to the mean of unstimulated transfected controls (untreated column) and were obtained in multiple experiments It is representative of the results. In RAW 264.7 cells expressing dominant negative MyD88lpr, κB-dependent luciferase activity induced by CpG oligonucleotide treatment, MPL or LPS positive control (FIG. 2A) was inhibited. The degree of inhibition of luciferase activity by dominant negative MyD88 was dependent on the amount of MyD88lpr plasmid used in the transfection (FIG. 2A) and the resulting level of MyD88lpr expression (data not shown). The indirect effects of CpG oligonucleotides mediated by induced expression of IL-18 or IL-1 (these receptors require MyD88 for signal transduction) may explain these results. However, in addition to the negative results obtained when RAW 264.7 macrophages were treated with recombinant IL-18 (see above), active IL-18, respectively, was generated by RAW264 by immunoblotting or ELISA. It was not detectable in the 7 cell extract or in the supernatant (data not shown).
[0062]
The dependence of κB-luciferase activation and MyD88lpr inhibition on methylation status and CG dinucleotide sequence in CpG oligonucleotides was investigated. Control oligonucleotide with GC replacing CG sequence (GC oligo) or control oligonucleotide synthesized with methyl-C instead of C (methyl CG) transfected with κB-Luc alone or with MyD88lpr RAW 264.7 cells. Referring to FIG. 2B, RAW 264.7 macrophages were transfected with κB-Luc alone (black bar) or κB-Luc + MyD88lpr (white bar) (1: 1 plasmid ratio); total DNA concentration was determined using an empty vector plasmid. Keeped constant by addition. Transfected cells were treated with an oligonucleotide containing an unmethylated CG motif (4 μM), an unmethylated GC (GC) or a similar oligonucleotide with a methylated CG motif (mCG) replacing the CpG motif, or a CG motif. Treated with the missing extraneous sequence (non-CpG) the next day for 6 hours. Results shown are the mean of duplicate wells +/− standard deviation normalized to the mean of unstimulated transfected controls (“untreated” column) and obtained in two experiments. Of the results obtained. As shown in FIG. 2B, methylation of CG sequences or their replacement with GC abolished almost all activation of κB-Luc by this oligonucleotide. Basal luciferase activity in cells treated with oligonucleotides lacking the CpG motif was not inhibited by MyD88lpr expression. These results suggest that MyD88-dependent receptors activated by CpG oligonucleotides have the expected specificity for unmethylated CG sequences that are intended to mimic bacterial DNA.
[0063]
In another set of experiments, three MyD88 constructs lacking the ability to transduce signals were amplified by PCR and placed under constitutive control of the CMV promoter (pCMV-FLAG vector, Sigma Aldrich). The first construct MyD88lpr contains a point mutation F56N in the cell death domain. The second construct MyD88-ΔDD is completely devoid of the cell death domain. The third construct MyD88-THD lacks both the cell death domain and the intermediate domain. DNA was transfected into RAW 264.7 cells or mouse embryonic NIH-3T3 cells using a cationic lipid reagent. The luciferase reporter plasmid was cotransfected. Processing of upstream κB-dependent promoter. Luciferase transcription relies on the release of NF-κB. Release of NF-κB is one of the final events in the Toll signaling pathway. Cells were then stimulated with CpG oligonucleotides and several control cytokines. Luciferase expression was assayed with a luminometer to determine whether the mutated MyD88 protein affected the CpG signal. Restriction analysis and sequencing results confirmed the presence of the MyD88 construct (data not shown). Dominant negative MyD88 specifically and selectively blocks signaling in response to CpG oligonucleotide stimulation (FIGS. 3A and 3B). This construct did not affect signaling through the TNF MyD88-dependent pathway (negative control), but inhibited MyD88-dependent LPS signaling and IL-1 signaling (FIG. 3A). The MyD88 TIR domain alone was sufficient to block signal transduction.
[0064]
These results demonstrate that MyD88 function is required for full activation of NF-κB by CpG oligonucleotides and MPL. Since all known receptors that require MyD88 share a common structural feature THD, this finding also implied that putative CpG receptor components have THD. The presence of receptors with THD and the involvement of Toll pathway components in signaling induced by the CpG motif is novel.
[0065]
This result provides a first insight into similar structural features of CpG receptor components. It will be appreciated by those skilled in the art that knowing that the immune response to an oligonucleotide containing an unmethylated CpG motif is mediated through receptors containing THD provides a new approach to identifying the corresponding gene. An investigation of the methylation dependence and good sequence specificity of the MyD88-dependent effects of CpG oligonucleotides and the relevance of these findings to other nucleotide-based activators of APC can be performed.
[0066]
(Example 4: TLR4 is not required for CpG signaling)
A preferred method for determining the polypeptide sequence of CpG-R is to test whether a known TLR can be a promising candidate. The requirement for TLR4 for response to LPS in mice has been demonstrated using the endotoxin non-responsive line C3H / HeJ with a point mutation in TLR4, and C57Bl / 10ScCr mice and C57Bl / 10ScNCr mice that do not express TLR4. (Vogel et al., J. Immunol., 1999, 162, 5666-70, Qureshi et al., J. Exp. Med., 1999, 189, 615-25, Chow et al., J. Biol. Chem., 1999, 274, 10687. -92, Hoshino et al., J. Immunol., 1999, 162, 3749-52 and Poltrak et al., Science, 1998, 282, 2085-8).
[0067]
To assess the role of TLR4 in the response of APC to CpG oligonucleotides and MPL in vitro, BMDDCs from LPS responsive mice (Balb / c) and C3H / HeJ mice were cultured overnight with these adjuvants, The upregulation of DC activation / maturation markers was then assayed. Cell surface CD86 expression was quantified by flow cytometry. CD86 is an NF-κB target gene that is upregulated in activated dendritic cells and macrophages. This enhances the ability to activate antigen-specific T cells. Referring to FIG. 4, BMDDCs from wild type (Balb / c; black bars) and TLR4 mutant mice (C3H / HeJ; white bars) were grown in GM-CSF for 6 days as described above, and then CpG Treated overnight with oligonucleotide (5 μM), control oligonucleotide (5 μM) or LPS (1 μg / ml) or left unstimulated. Cell surface expression of CD86 on live CD11c positive cells was assayed by flow cytometry. Results shown are MFs of BMDDC treated with adjuvant, normalized to geometric mean fluorescence (MF) of untreated BMDDC from the same culture, and are representative of results obtained in three experiments. is there. As shown in FIG. 4, wild-type BMDDC treated with CpG oligonucleotide, LPS showed increased cell surface CD86 expression. In TLR4 mutant BMDDC, no change in CD86 expression is observed in MPL-treated or LPS-treated cells, while an increase in CD86 expression similar to that observed in wild-type BMDDC is seen in CpG oligonucleotide-treated cultures. (Figure 4 and data not shown). Thus, TLR4 is required for in vitro APC activation by LPS and MPL, but not for in vitro APC activation by CpG oligonucleotides.
[0068]
Additional experiments similar to those described above can be performed to test other known Toll-like receptors to better characterize the exact nature of CpG-R. This can be done in any comparable cell line with known intact TLRs and mutations that render these receptors non-functional. Identification of cells with a receptor that is activated by CpG when intact but not activated when the receptor is deficient pinpoints exactly which TLR is present and which particular THD is present .
[0069]
The results disclosed herein also provide a useful approach to develop compounds that can act as agonists or antagonists of cellular signaling mediated by receptors that require the adapter protein MyD88 for the signaling pathway. It will be appreciated by those skilled in the art to provide. Compounds can be incubated with cells and assayed to see if a cascade of events known to follow from CpG activation has resulted. The cells can then be transfected with the MyD88lpr gene. Compounds that produce an effect in controls without the MyD88lpr gene but do not produce an effect when the MyD88lpr gene is expressed have been shown to activate intracellular signaling through pathways that require the MyD88 adapter protein. .
[0070]
(Example 5: Isolation of CpG-R)
A number of different procedures can be utilized to identify Toll-related CpG-R components. First, one skilled in the art can examine the ability of known and novel Toll-like receptors to confer responsiveness to CpG oligonucleotides to non-responsive cells. Second, one of skill in the art can use MyD88 to purify interacting proteins that are specifically activated by CpG oligonucleotides and not by other Toll receptor ligands (eg, LPS), Alternatively, the corresponding cDNA can be isolated using a yeast two-hybrid system.
[0071]
Mouse Toll-like receptors 1-6 (TLR1-6) have closely related human homologs. Full or partial sequences are available in public databases for mouse genes. An expression vector encoding a TLR can be transfected into a CpG non-responsive cell line (eg, NIH3T3) with a reporter gene activated by a CpG oligonucleotide (eg, κB-luciferase). If any of the co-transfected TLRs confer responsiveness to the CpG oligonucleotide, this is reflected by expression of the reporter gene upon treatment with the CpG oligonucleotide.
[0072]
A similar approach can be used to test the involvement of any candidate CpG-R component in CpG signaling, including novel TLRs. Approaches for identifying and cloning novel Toll-related receptors include genomics, sequence database screening, degenerate PCR, yeast two-hybrid systems, and library screening by hybridization. Active CpG oligonucleotides are best described in rodents, not primates, so they are studied in mouse systems to identify Toll-related CpG components and then using human homologs, for example using PCR Is preferably isolated.
[0073]
MyD88lpr contains an intact Toll homology domain and a single inactivation point mutation within the cell death domain and can be expressed as a recombinant protein in either bacterial or eukaryotic cells. Recombinant proteins can be used as affinity reagents to isolate and purify interacting proteins from CpG oligonucleotide treated cells (eg, RAW 264.7). Binding of downstream effector molecules (eg, IRAK, TRAF6) in the TLR signaling pathway should be minimized or eliminated by “lpr” mutations in the cell death domain. Thus, a protein that specifically binds to MyD88lpr is a protein that interacts with THD and / or a region that links THD to the cell death domain. Since Toll homology domains associate via homologous interactions, CpG-R components containing THD are expected to bind to MyD88 THD. Once the putative CpG-R is affinity purified for MyD88, the corresponding cDNA can be isolated by standard methods such as, for example, peptide analysis and PCR.
[0074]
Similarly, MyD88lpr can be used as a bait in a yeast two-hybrid system to isolate cDNA encoding interacting proteins. The relevance of these cDNAs to CpG oligonucleotide-induced signaling can then be tested as described above.
[Brief description of the drawings]
FIG. 1A is a bar graph showing activation of NF-κB in RAW 264.7 macrophages. Here, RAW 264.7 cells were transfected with a luciferase reporter gene driven by κB, and after 24 hours interleukin-1 (IL-1; 20 ng / ml), MPL (1 μg / ml), LPS (1 μg / ml). ml) or phosphorothioate CpG oligonucleotide (4 μM) for 6 hours.
FIG. 1B is a graph showing CpG oligonucleotide-induced κB-luciferase (κB-Luc) activation. Here, RAW 264.7 cells were transiently transfected with κB-luciferase reporter plasmid and phosphorothioate oligonucleotides (0.25-8 μM) or control containing the CpG motif at the indicated concentrations after 24 hours. Treated with oligonucleotide (4 μM) for 6 hours.
FIG. 1C is a graph showing the kinetics of κB-luciferase activation by CpG oligonucleotides or LPS. Here, RAW 264.7 cells were transiently transfected with κB-Luc and treated 24 hours later with CpG oligonucleotides (4 μM; open circles) or LPS (1 μg / ml; filled circles).
FIG. 2A is a graph showing that the dominant negative mutant MyD88 (MyD88lpr) inhibits CpG oligonucleotide-induced κB-luciferase activation. Here, RAW 264.7 cells were transformed with κB-Luc (1 μg) alone (black bar) or plasmid encoding MyD88lpr (in two ratios: 10: 1 (gray bar) or 1: 1 (white bar)). Transfected.
FIG. 2B is a bar graph showing that NF-κB activation and inhibition by MyD88lpr are dependent on unmethylated CG sequences. Here, RAW 264.7 macrophages were transfected with κB-Luc alone (black bar), or κB-Luc and MyD88lpr (white bar) (1: 1 plasmid ratio).
FIG. 3A is a bar graph showing a representative luciferase assay of NIH-3T3 cells transfected with a plasmid encoding MyD88 dominant negative construct.
FIG. 3B is a bar graph showing a representative luciferase assay of RAW 264.7 cells transfected with a plasmid encoding MyD88 dominant negative construct.
FIG. 4 is a bar graph showing Toll-like receptor 4 (TLR4) independence on APC response to CpG oligonucleotide treatment. Here, immature bone marrow derived dendritic cells (BMDCC) derived from wild type (Balb / c; black bars) and TLR4 mutant mice (CH3 / HeJ; white bars) were obtained in GM-CSF as described above. Grown daily and then treated overnight with CpG oligonucleotide (5 μM), control oligonucleotide (5 μM), LPS (1 μg / ml) or left unstimulated.
[Sequence Listing]

Claims (9)

  Use of a polypeptide as a CpG receptor, wherein the polypeptide comprises a Toll homology domain and binds to a CpG oligonucleotide.   2. Use according to claim 1, wherein the polypeptide is capable of interacting with MyD88 protein.   3. Use according to claim 1 or claim 2, wherein the polypeptide is a Toll-like receptor (TLR).   A method of identifying a compound that binds to or modulates the activity of a CpG receptor, wherein the receptor comprises a Toll homology domain and binds to a CpG oligonucleotide, the method comprising the receptor or Contacting a cell expressing the receptor with a compound; and whether the compound binds to the receptor or whether the compound modulates the ability of the receptor to bind to a CpG oligonucleotide. Determining the method. 5. The method of claim 4 , wherein the polypeptide can interact with MyD88 protein. 6. A method according to claim 4 or claim 5 , wherein the polypeptide is a Toll-like receptor (TLR).   A receptor / ligand complex, wherein the receptor is a polypeptide comprising a Toll homology domain and the ligand is a CpG oligonucleotide. 8. The complex according to claim 7 , wherein the polypeptide is capable of interacting with MyD88 protein. 9. The complex according to claim 7 or claim 8 , wherein the polypeptide is a Toll-like receptor (TLR).

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